Oxydans Cells Research Articles

A facile process for adipic acid production in high yield by oxidation of 1,6-hexanediol using the resting cells of Gluconobacter oxydans

BackgroundAdipic acid (AA) is one of the most important industrial chemicals used mainly for the production of Nylon 6,6 but also for making polyurethanes, plasticizers, and unsaturated polyester resins, and more recently as a component in the biodegradable polyester poly(butylene adipate terephthalate) (PBAT). The main route for AA production utilizes benzene as feedstock and generates copious amounts of the greenhouse gas NO2. Hence, alternative clean production routes for AA from renewable bio-based feedstock are drawing increasing attention. We have earlier reported the potential of Gluconobacter oxydans cells to oxidize 1,6-hexanediol, a potentially biobased diol to AA.ResultsThe present report involves a study on the effect of different parameters on the microbial transformation of 1,6-hexanediol to adipic acid, and subsequently testing the process on a larger lab scale for achieving maximal conversion and yield. Comparison of three wild-type strains of G. oxydans DSM50049, DSM2003, and DSM2343 for the whole-cell biotransformation of 10 g/L 1,6-hexanediol to adipic acid in batch mode at pH 7 and 30 °C led to the selection of G. oxydans DSM50049, which showed 100% conversion of the substrate with over 99% yield of adipic acid in 30 h. An increase in the concentrations of the substrate decreased the degree of conversion, while the product up to 25 g/L in batch and 40 g/L in fed-batch showed no inhibition on the conversion. Moreover, controlling the pH of the reaction at 5–5.5 was required for the cascade oxidation reactions to work. Cell recycling for the biotransformation resulted in a significant decrease in activity during the third cycle. Meanwhile, the fed-batch mode of transformation by intermittent addition of 1,6-hexanediol (30 g in total) in 1 L scale resulted in complete conversion with over 99% yield of adipic acid (approximately 37 g/L). The product was recovered in a pure form using downstream steps without the use of any solvent.ConclusionA facile, efficient microbial process for oxidation of 1,6-hexanediol to adipic acid, having potential for scale up was demonstrated. The entire process is performed in aqueous medium at ambient temperatures with minimal greenhouse gas emissions. The enzymes involved in catalyzing the oxidation steps are currently being identified.

Gluconobacter Oxydans-Based MFC with PEDOT:PSS/Graphene/Nafion Bioanode for Wastewater Treatment.

Microbial fuel cells (MFCs) are a variety of bioelectrocatalytic devices that utilize the metabolism of microorganisms to generate electric energy from organic matter. This study investigates the possibility of using a novel PEDOT:PSS/graphene/Nafion composite in combination with acetic acid bacteria Gluconobacter oxydans to create a pure culture MFC capable of effective municipal wastewater treatment. The developed MFC was shown to maintain its activity for at least three weeks. The level of COD in municipal wastewater treatment was reduced by 32%; the generated power was up to 81 mW/m2 with a Coulomb efficiency of 40%. Combining the MFC with a DC/DC boost converter increased the voltage generated by two series-connected MFCs from 0.55 mV to 3.2 V. A maximum efficiency was achieved on day 8 of MFC operation and was maintained for a week; capacitors of 6800 µF capacity were fully charged in ~7 min. Thus, G. oxydans cells can become an important part of microbial consortia in MFCs used for treatment of wastewaters with reduced pH.

Direct Bioelectrocatalytic Oxidation of Glucose by Gluconobacter oxydans Membrane Fractions in PEDOT:PSS/TEG-Modified Biosensors.

Recent years have witnessed an ever-increasing interest in developing electrochemical biosensors based on direct electron transfer-type bioelectrocatalysis. This work investigates the bioelectrocatalytic oxidation of glucose by membrane fractions of Gluconobacter oxydans cells on screen-printed electrodes modified with thermally expanded graphite and poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS). Electrooxidation of glucose was shown to occur without the presence of electron transport mediators. Chronoamperometric and cyclic voltametric characteristics showed an increase of anodic currents at electrode potentials of 0–500 mV relative to the reference electrode (Ag/AgCl). The direct electron transfer effect was observed for non-modified PEDOT:PSS as well as for PEDOT:PSS linked with crosslinkers and conductive fillers such as polyethylene glycol diglycidyl or dimethyl sulfoxide. Bioelectrodes with this composite can be successfully used in fast reagent-free glucose biosensors.

Perspective of Using Gluconacetobacter sucrofermentas VKPM B-11267 in Biofuel Cells

The bioelectrochemical and spectral properties of immobilized Gluconacetobacter sucrofermentas VKPM B-11267 bacteria were studied in the presence and absence of multiwalled carbon nanotubes (MWCNTs). The obtained characteristics were compared with the characteristics of Gluconobacter oxydans, which are phylogenetically close to them and are widely used in bioelectrochemistry. It was shown that modification of the bioelectrode with carbon nanotubes leads to a significant increase in the current level (by 2.5–3 times), as well as to a decrease in the total resistance both in the absence of substrates and in their presence. The potential use of immobilized G. sucrofermentas cells as part of a microbial fuel cell (MFC) was considered. The specific electrical power of an MFC based on immobilized G. sucrofermentas cells was lower than that of an MFC based on G. oxydans cells. Nevertheless, the results obtained indicate that G. sucrofermentas VKPM B-11267 cells can serve as a biocatalyst in MFCs.

The Auxiliary NADH Dehydrogenase Plays a Crucial Role in Redox Homeostasis of Nicotinamide Cofactors in the Absence of the Periplasmic Oxidation System in Gluconobacter oxydans NBRC3293.

Gluconobacter oxydans has the unique property of a glucose oxidation system in the periplasmic space, where glucose is oxidized incompletely to ketogluconic acids in a nicotinamide cofactor-independent manner. Elimination of the gdhM gene for membrane-bound glucose dehydrogenase, the first enzyme for the periplasmic glucose oxidation system, induces a metabolic change whereby glucose is oxidized in the cytoplasm to acetic acid. G. oxydans strain NBRC3293 possesses two molecular species of type II NADH dehydrogenase (NDH), the primary and auxiliary NDHs that oxidize NAD(P)H by reducing ubiquinone in the cell membrane. The substrate specificities of the two NDHs are different from each other: primary NDH (p-NDH) oxidizes NADH specifically but auxiliary NDH (a-NDH) oxidizes both NADH and NADPH. We constructed G. oxydans NBRC3293 derivatives defective in the ndhA gene for a-NDH, in the gdhM gene, and in both. Our ΔgdhM derivative yielded higher cell biomass on glucose, as reported previously, but grew at a lower rate than the wild-type strain. The ΔndhA derivative showed growth behavior on glucose similar to that of the wild type. The ΔgdhM ΔndhA double mutant showed greatly delayed growth on glucose, but its cell biomass was similar to that of the ΔgdhM strain. The double mutant accumulated intracellular levels of NAD(P)H and thus shifted the redox balance to reduction. Accumulated NAD(P)H levels might repress growth on glucose by limiting oxidative metabolisms in the cytoplasm. We suggest that a-NDH plays a crucial role in redox homeostasis of nicotinamide cofactors in the absence of the periplasmic oxidation system in G. oxydansIMPORTANCE Nicotinamide cofactors NAD+ and NADP+ mediate redox reactions in metabolism. Gluconobacter oxydans, a member of the acetic acid bacteria, oxidizes glucose incompletely in the periplasmic space-outside the cell. This incomplete oxidation of glucose is independent of nicotinamide cofactors. However, if the periplasmic oxidation of glucose is abolished, the cells oxidize glucose in the cytoplasm by reducing nicotinamide cofactors. Reduced forms of nicotinamide cofactors are reoxidized by NADH dehydrogenase (NDH) on the cell membrane. We found that two kinds of NDH in G. oxydans have different substrate specificities: the primary enzyme is NADH specific, and the auxiliary one oxidizes both NADH and NADPH. Inactivation of the latter enzyme in G. oxydans cells in which we had induced cytoplasmic glucose oxidation resulted in elevated intracellular levels of NAD(P)H, limiting cell growth on glucose. We suggest that the auxiliary enzyme is important if G. oxydans grows independently of the periplasmic oxidation system.

Repeated production of 6-(N-hydroxyethyl)-amino-6-deoxy-α-L-sorbofuranose by immobilized Gluconobacter oxydans cells with a strategy of in situ exhaustive cell regeneration.

The major troubles in 6-(N-hydroxyethyl)-amino-6-deoxy-α-L-sorbofuranose (6NSL) production from N-2-hydroxyethyl glucamine (NHEG) by Gluconobacter oxydans were low cell yield during cell preparation and loss of cells' biocatalytic ability during biotransformation, resulting in high production cost and low 6NSL production. The target of this work was to enhance 6NSL production by reusing cells and improving the cells biocatalytic ability. First, inhibitory effects of substrate and product on 6NSL production, and optimization of cell regeneration condition were investigated, respectively. Then repeated production of 6NSL by immobilized cell using a strategy of in situ exhaustive cell regeneration in a bubble column bioreactor was developed. As a result, the bioprocess underwent nine cycles, the average 6NSL production and conversion rate of NHEG to 6NSL reached 42.6gL-1 and 83.1% in each batch was achieved, respectively.

STIMULATING EFFECTS OF MULTIWALLED CARBON NANOTUBES ON IMMOBILIZED GLUCONACETOBACTER SUCROFERMENTAS VKPM B-11267 CELLS

The Gluconacetobacter sucrofermentas bacterial cells are immobilized in a chitosan gel on the surface of an electrode obtained by matrix printing. The nature of the change in the bioelectrochemical parameters of the immobilized bacteria under the influence of multiwalled carbon nanotubes (MWCNTs) are studied. We studied the electrochemical signals of cells upon the introduction of substrates (glucose, ethanol) by three methods: chronoamperometry, cyclic voltammetry, and impedance spectroscopy. The introduction of ethyl alcohol at a concentration of 1 mM leads to a decrease in the total resistance of the electrode by a factor of ~40. Modification of the electrode using MWCNTs leads to an increase in the current (by factors of 8 to 10) and an additional decrease in the total electrode resistance by a factor of ~10. These effects are similar to the effects of substrates and MWCNTs on the phylogenetically related Gluconobacter oxydans cells. The formation of bacterial cellulose (BC) membrane cells during the growth does not fundamentally change the effects of the substrates and MWCNTs.

Valorization of Waste Glycerol to Dihydroxyacetone with Biocatalysts Obtained from Gluconobacter oxydans

Waste glycerol is the main by-product generated during biodiesel production, in an amount reaching up to 10% of the produced biofuel. Is there any method which allows changing this waste into industrial valuable compounds? This manuscript describes a method for valorization of crude glycerol via microbial bioconversion. It has been shown that the use of free and immobilized biocatalysts obtained from Gluconobacter oxydans can enable beneficial valorization of crude glycerol to industrially valuable dihydroxyacetone. The highest concentration of this compound, reaching over 20 g·L−1, was obtained after 72 h of biotransformation with free G. oxydans cells, in a medium containing 30 or 50 g·L−1 of waste glycerol. Using a free cell extract resulted in higher concentrations of dihydroxyacetone and a higher valorization efficiency (up to 98%) compared to the reaction with an immobilized cell extract. Increasing waste glycerol concentration to 50 g·L−1 causes neither a faster nor higher increase in product yield and reaction efficiency compared to its initial concentration of 30 g·L−1. The proposed method could be an alternative for utilization of a petrochemical waste into industry applicated chemicals.

Batch and Repeated-Batch Fermentation for 1,3-Dihydroxyacetone Production from Waste Glycerol Using Free, Immobilized and Resting Gluconobacter oxydans Cells

Conversion of biodiesel derived waste/crude glycerol to higher value products is a potential way for enhancing the economy of biodiesel industry. Among several products, dihydroxyacetone (DHA) is one of the fine chemicals known to be used in pharmaceutical and cosmetic industry. However, microbial DHA production by Gluconobacter oxydans is inhibited by high concentration of substrate as well as product. Therefore, the present study is focused on DHA production by batch and repeated-batch fermentation to obviate the substrate inhibition effect. Apart from using free cells for fermentation experiments, this study is more focused towards use of immobilized and resting G. oxydans cells with pure/crude glycerol as substrate. For batch experiment with immobilized cells, a final DHA concentration of 17.83 g/L was observed in presence of pure glycerol, which was approx. ninefold higher than free and resting cells experiments. Repeated-batch experiment with four times crude glycerol feeding (10 g/L each time) resulted a DHA concentration of 35.95 g/L, with 89.88% conversion rate within 96 h of fermentation. Increase in initial glycerol feed and final DHA concentration in the fermentation broth, decreased the value of rate constant (k1), which further corroborate the substrate and product inhibition effect.

Three-Dimensional Organization of Self-Encapsulating Gluconobacter oxydans Bacterial Cells.

Self-organized bacteria have been the subject of interest for a number of applications, including the construction of microbial fuel cells. In this paper, we describe the formation of a self-organized, three-dimensional network that is constructed using Gluconobacter oxydans B-1280 cells in a hydrogel consisting of poly(vinyl alcohol) (PVA) with N-vinyl pyrrolidone (VP) as a cross-linker, in which the bacterial cells are organized in a particular side-by-side alignment. We demonstrated that nonmotile G. oxydans cells are able to reorganize themselves, transforming and utilizing PVA–VP polymeric networks through the molecular interactions of bacterial extracellular polysaccharide (EPS) components such as acetan, cellulose, dextran, and levan. Molecular dynamics simulations of the G. oxydans EPS components interacting with the hydrogel polymeric network showed that the solvent-exposed loops of PVA–VP extended and engaged in bacterial self-encapsulation.

Process optimization and analysis of product inhibition kinetics of crude glycerol fermentation for 1,3-Dihydroxyacetone production

In present study, statistical optimization of biodiesel–derived crude glycerol fermentation to DHA by immobilized G. oxydans cells over polyurethane foam is reported. Effect of DHA (product) inhibition on crude glycerol fermentation was analyzed using conventional biokinetic models and new model that accounts for both substrate and product inhibition. Optimum values of fermentation parameters were: pH=4.7, temperature=31°C, initial substrate concentration=20g/L. At optimum conditions, DHA yield was 89% (17.83g/L). Effect of product inhibition on fermentation was trivial for DHA concentrations ≤30g/L. At higher concentrations (≥50g/L), kinetics and yield of fermentation showed marked reduction with sharp drop in Vmax and KS values. Inhibition effect was more pronounced for immobilized cells due to restricted transport of fermentation mixture across polyurethane foam. Retention of fermentation mixture in immobilized matrix resulted in higher localized DHA concentration that possibly enhanced inhibition effect.

Utilization of a waste glycerol fraction using and reusing immobilized Gluconobacter oxydans ATCC 621 cell extract

Abstract Background Depletion of petroleum resources has enforced the search for alternative sources of renewable energy. Introduction of biofuels into the market was expected to become a solution to this disadvantageous situation. Attempts to cover fuel demand have, however, caused another severe problem—the waste glycerol generated during biodiesel production at a concentration of approximately 10% w/w. This, in turn, prompted a global search for effective methods of valorization of the waste fraction of glycerol. Results Utilization of the waste fraction at 48 h with an initial glycerol concentration of 30 g·L -1 and proceeding with 62% efficiency enabled the production of 9 g·L -1 dihydroxyacetone at 50% substrate consumption. The re-use of the immobilized biocatalyst resulted in a similar concentration of dihydroxyacetone (8.7 g·L -1 ) in two-fold shorter time, with an efficiency of 85% and lower substrate consumption (35%). Conclusions The method proposed in this work is based on the conversion of waste glycerol to dihydroxyacetone in a reaction catalyzed by immobilized Gluconobacter oxydans cell extract with glycerol dehydrogenase activity, and it could be an effective way to convert waste glycerol into a valuable product.

Kinetic analysis of dihydroxyacetone production from crude glycerol by immobilized cells of Gluconobacter oxydans MTCC 904

The present study has investigated kinetic features of bioconversion of biodiesel-derived crude glycerol to dihydroxyacetone with immobilized Gluconobacter oxydans cells using modified Haldane substrate-inhibition model. The results have been compared against free cells and pure glycerol. Relative variations in the kinetic parameters KS, KI, Vmax, n and X reveal that immobilized G. oxydans cells (on PU foam substrate) with crude glycerol as substrate give higher order of inhibition (n) and lower maximum reaction velocities (Vmax). These results are essentially implications of substrate transport restrictions across immobilization matrix, which causes retention of substrate in the matrix and reduction in fractional available substrate (X) for the cells. This causes reduction in both KS (substrate concentration at Vmax/2) and KI (inhibition constant) as compared to free cells. For immobilized cells, substrate concentration (Smax) corresponding to Vmax is practically same for both pure and crude glycerol as substrate.

Vinegar Production from Jabuticaba (Myrciaria jaboticaba) Fruit Using Immobilized Acetic Acid Bacteria.

Cell immobilization comprises the retention of metabolically active cells inside a polymeric matrix. In this study, the production of jabuticaba (Myrciaria jaboticaba) vinegar using immobilized Acetobacter aceti and Gluconobacter oxydans cells is proposed as a new method to prevent losses of jabuticaba fruit surplus. The pulp of jabuticaba was processed and Saccharomyces cerevisiae CCMA 0200 was used to ferment the must for jabuticaba wine production. Sugars, alcohols (ethanol and glycerol) and organic acids were assayed by high-performance liquid chromatography. Volatile compounds were determined by gas chromatography-flame ionization detector. The ethanol content of the produced jabuticaba wine was approx. 74.8 g/L (9.5% by volume) after 168 h of fermentation. Acetic acid fermentation for vinegar production was performed using a mixed culture of immobilized A. aceti CCT 0190 and G. oxydans CCMA 0350 cells. The acetic acid yield was 74.4% and productivity was 0.29 g/(L·h). The vinegar had particularly high concentrations of citric (6.67 g/L), malic (7.02 g/L) and succinic (5.60 g/L) acids. These organic acids give a suitable taste and flavour to the vinegar. Seventeen compounds (aldehydes, higher alcohols, terpene, acetate, diether, furans, acids, ketones and ethyl esters) were identified in the jabuticaba vinegar. In conclusion, vinegar was successfully produced from jabuticaba fruits using yeast and immobilized mixed cultures of A. aceti and G. oxydans. To the best of our knowledge, this is the first study to use mixed culture of immobilized cells for the production of jabuticaba vinegar.

Biooxidation of 2-phenylethanol to phenylacetic acid by whole-cell Gluconobacter oxydans biocatalyst immobilized in polyelectrolyte complex capsules

A high-performance biocatalyst in the form of encapsulated cells of Gluconobacter oxydans have been developed for production of phenylacetic acid (PAA) as a natural flavor component. Polyelectrolyte complex (PEC) capsules consisting of sodium alginate, cellulose sulfate, poly(methylene-co-guanidine), CaCl2, and NaCl were used for highly controlled and mild encapsulation of cells. Utilization of encapsulated G. oxydans cells was a significant improvement on existing data on operational stability of cells and cumulative product concentration during biocatalytic production of PAA from 2-phenylethanol. Concerning operational stability, encapsulated cells were active over 12 cycles with a high biotransformation rate, while free cells were inactive after 7 cycles of use. The biocatalytic properties of encapsulated G. oxydans were tested in a bubble column reactor over 7 days with a final cumulative product concentration of 25 g/L. High cell viability (90%) was observed within PEC capsules by confocal laser scanning microscopy, performed before and after repetitive PAA production in the bubble column reactor. The surface microstructure of fully hydrated capsules with and without G. oxydans cells was investigated and compared using an environmental scanning electron microscope.

Gluconobacter sp. cells for manufacturing of effective electrochemical biosensors and biofuel cells

AbstractGluconobacter oxydans bacteria exhibit a unique metabolism for quick and incomplete oxidation of a wide range of different compounds (aldoses, ketoses, mono- and poly-alcohols, etc.). Such biotransformation efficiency with simple biomass production led to the industrial applications of these bacteria in the production of several important commodities. Their respiratory activity can also be successfully studied and used in the field of bioelectrochemistry. The main aim of this review is to present various strategies to improve selectivity of assays using intact/treated cells of G. oxydans, to introduce the application of G. oxydans-based biosensors in selective monitoring of analytes during biotransformation processes and to provide information about utilizable sugars in fermentation media or in biological oxygen demand value determination. The final part of the review describes potential application of G. oxydans cells in the generation of electricity from complex fuels within microbial fuel cells by advanced direct electron transfer route between bacterial cells and electrodes.

Modified Gold Surfaces with Gold Nanoparticles and 6‐(Ferrocenyl)hexanethiol: Design of a Mediated Microbial Sensor

AbstractHerein, a mediated Gluconobacter oxydans biosensor was constructed by immobilizing the intact cells on the modified gold electrode. The surface was initially modified with gold nanoparticles and then, self‐assembled monolayers were formed by using 6‐(ferrocenyl) hexanethiol. Finally, G. oxydans cells were adsorbed onto the surfaces and the bio‐active layer was covered with a dialysis membrane. As well as the response characteristics, stabilities and substrate specificities ere investigated.

A novel functional conducting polymer as an immobilization platform

Here, we present the fabrication of conducting polymer based enzymatic and microbial biosensors. To obtain immobilization platforms for both pyranose oxidase (PyOx) and Gluconobacter oxydans, the graphite electrode surface was modified with the polymer of 4-amino-N-(2,5-di(thiophen-2-yl)-1H-pyrrol-1-yl)benzamide (HKCN) which has free amino groups on the surface for further bioconjugation reactions with the biomolecules. Initially, the electrode surface was covered with HKCN via electropolymerization. Then, either PyOx or G. oxydans cell was stabilized using glutaraldehyde as a cross-linker. After optimization of biosensors, analytical characterization and surface imaging studies were investigated. The change of current depends on glucose concentration between 0.05–1.0mM and 0.25–2.5mM with HKCN/PyOx and HKCN/G. oxydans biosensors in batch systems. Also, the calibration graphs were obtained for glucose in FIA mode, and in this case, linear ranges were found to be 0.01–1.0mM and 0.1–7.5mM for HKCN/PyOx and HKCN/G. oxydans, respectively.

Overexpression of a type II 3-dehydroquinate dehydratase enhances the biotransformation of quinate to 3-dehydroshikimate in Gluconobacter oxydans

Shikimate and 3-dehydroshikimate are useful chemical intermediates for the synthesis of various compounds, including the antiviral drug oseltamivir. Here, we show an almost stoichiometric biotransformation of quinate to 3-dehydroshikimate by an engineered Gluconobacter oxydans strain. Even under pH control, 3-dehydroshikimate was barely detected during the growth of the wild-type G. oxydans strain NBRC3244 on the medium containing quinate, suggesting that the activity of 3-dehydroquinate dehydratase (DHQase) is the rate-limiting step. To identify the gene encoding G. oxydans DHQase, we overexpressed the gox0437 gene from the G. oxydans strain ATCC621H, which is homologous to the aroQ gene for type II DHQase, in Escherichia coli and detected high DHQase activity in cell-free extracts. We identified the aroQ gene in a draft genome sequence of G. oxydans NBRC3244 and constructed G. oxydans NBRC3244 strains harboring plasmids containing aroQ and different types of promoters. All recombinant G. oxydans strains produced a significant amount of 3-dehydroshikimate from quinate, and differences between promoters affected 3-dehydroshikimate production levels with little statistical significance. By using the recombinant NBRC3244 strain harboring aroQ driven by the lac promoter, a sequential pH adjustment for each step of the biotransformation was determined to be crucial because 3-dehydroshikimate production was enhanced. Under optimal conditions with a shift in pH, the strain could efficiently produce a nearly equimolar amount of 3-dehydroshikimate from quinate. In the present study, one of the important steps to convert quinate to shikimate by fermenting G. oxydans cells was investigated.

Amine intercalated clay surfaces for microbial cell immobilization and biosensing applications

Trimethylamine (TM) intercalated montmorillonite (Mont) clay was prepared and characterized using X-ray diffraction, Fourier transform infrared spectroscopy, zeta potential and thermal gravimetric measurements. Gluconobacter oxydans cells were immobilized on the micro-structured matrix and used in microbial sensor applications. Sensor responses were based on the respiratory activity of the cells and the consumption of oxygen was monitored at −0.7 V (vs. Ag/AgCl and platinum electrodes) by using glucose as a substrate. Stabilization of the bacteria was performed on the Mont using glutaraldehyde. Optimization of the TM-Mont/G. oxydans sensor and examination of the electrochemical mechanism were carried out in a batch system. Measurements concerning analytical characteristics, operational stability, repeatability and substrate specificity depending on the carbon source in the culture were investigated in a flow injection analysis (FIA) system. Linear ranges were found between 0.15 and 5.0 mM for the batch mode and 0.1 and 5.0 mM for the FIA system, respectively. Finally, real samples were analyzed and were compared with the results of a spectrophotometric method as reference.